Prevalence of Chromosomally Located blaCTX-M-55 in Salmonella Typhimurium ST34 Isolates Recovered from a Tertiary Hospital in Guangzhou, China

ABSTRACT Nontyphoidal Salmonella (NTS) is one of the most prevalent bacterial causes of gastrointestinal infections worldwide. Meanwhile, the detection rate of CTX-M-55 ESBL-positive has increased gradually in China. To identify the molecular epidemiological and genomic characteristics of blaCTX-M-55-carrying nontyphoidal Salmonella (NTS) clinical isolates, a total of 105 NTS isolates were collected from a Chinese tertiary hospital. Antimicrobial susceptibility testing was performed to determine the resistance phenotype. Whole-genome sequencing and bioinformatics analysis were used to determine the antimicrobial resistance genes, serotypes, phylogenetic relationships, and the genetic environment of the blaCTX-M-55 gene. The results showed that among the 22 ceftriaxone resistant isolates, the blaCTX-M-55 was the most common β-Lactamase gene carried by 14 isolates, including serotypes S. Typhimurium (10/14), S. Muenster (2/14), S. Rissen (1/14), and S. Saintpaul (1/14). Phylogenetic analysis shows that 10 blaCTX-M-55-positive S. Typhimurium ST34 isolates were divided into two clusters. The genetic relationship of isolates in each cluster was very close (≤10 cgMLST loci). The blaCTX-M-55 gene was located on the chromosome in 10 isolates, on IncI1 plasmid in three isolates, and IncHI2 plasmid in one isolate. In conclusion, the blaCTX-M-55 gene, mainly located on the chromosome of S. Typhimurium ST34 isolates, was the main driving force associated with the resistance of NTS to cephalosporins. Therefore, close attention to the clonal dissemination of blaCTX-M-55-carrying S. Typhimurium ST34 in clinical settings must be monitored carefully. IMPORTANCE ESCs are the first choice for treating NTS infections. However, ESBLs and AmpC β-lactamases are the most typical cause for ESCs resistance. The CTX-M-55 ESBL-positive rate has gradually increased in the clinic in recent years. At present, the research about blaCTX-M-55-positive Salmonella mainly focuses on the foodborne animals or the environment while less on clinical patients. Thus, this study was carried out for identifying molecular epidemiological and genomic characteristics of blaCTX-M-55-carrying NTS clinical isolates. The results showed that the blaCTX-M-55 gene, mainly located on the chromosome of S. Typhimurium ST34 isolates from Conghua District, was the main driving force associated with the resistance of NTS to cephalosporins. Therefore, our work highlights the importance of monitoring the clonal dissemination of blaCTX-M-55-carrying S. Typhimurium ST34 in clinical settings.

Authors, should consider publishing this work with another angle, as the number of strains does not reflect the title of the study. The text should be revised with regard to orthograph, examples: Line 79: space missing between clinic and (6) Line 112: E. coli should be in italic Line 130: space missing between 29 and "and" Line 132: space missing between server and (31) Line 133: 2 spaces between for and subsequent Reviewer #2 (Comments for the Author): This manuscript investigated the prevalence of blaCTX-M-55 gene in Non-typhoidal Salmonella from a tertiary hospital between May 2020 and February 2021, and clarified the blaCTX-M-55 gene mainly located on the chromosome of S. Typhimurium ST34, highlighting the importance of monitoring the clonal dissemination of blaCTX-M-55-carrying S. Typhimurium ST34 in clinical settings. In generally, the outcomes have clinical implications, but sample size is limited and further investigation is needed. The discussion should be more condensed and focused.
Major concerns: (1) The authors claimed that blaCTX-M-55 is located on chromosomes or plasmids in 14 strains. Did they do Pulsed-field gel electrophoresis with S1 nuclease and Southern blotting experiments? How else would they select three representative strains for Nanopore sequencing? Please provide the detail information and data.
(2) Line 271-273: This point here is not appropriate. How could you get the point? There have no IS26 in plasmid IncHI2.
(3) The Figures in this manuscript are too simple and crude to contain much useful information, and are poorly made.
Minor comments: (1) In line 32,33, 36, 37, there should be Spaces between words (2) In line 128, the FastQC reference may not be quite correct, please double-check it.
(3) In lines 131-132, what are the parameters in the process of determining resistance genes and plasmid replication subtypes through the CGE server, for similarity and coverage, etc.? These should be stated.
(4) In line 683, 684, 686 and 687. ISECP1, E.coli, Salmonella as well as resistance genes should be italicized. Such errors also appeared elsewhere in the manuscript, please check.
Reviewer #3 (Comments for the Author): This paper characterized 105 clinical Salmonella strains, the results should be more concentrate. For example, how many patients are the strains from? What's the meaning of the result of months and detection rate of male and female patients? This paper can be more important by just describe and discuss most important findings. Other problems are listed below. 1.Capitalize the first letter of the title? 2.Spaces are missing in many places 3.Line 112, the name of strain should be in Italic. 4.The results should be re-organized. It is better to have a separate result for antibiotic resistance genes in Line 188-195. 5.Phylogenetic analysis should contain more strains from China. 6.Many grammer problems. 7.Figure should be more clear.

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This manuscript investigated the prevalence of bla CTX-M-55 gene in Non-typhoidal
Salmonella from a tertiary hospital between May 2020 and February 2021, and clarified the bla CTX-M-55 gene mainly located on the chromosome of S. Typhimurium ST34, highlighting the importance of monitoring the clonal dissemination of blaCTX-M-55-carrying S. Typhimurium ST34 in clinical settings.
In generally, the outcomes have clinical implications, but sample size is limited and further investigation is needed. The discussion should be more condensed and focused.
Major concerns: (1) The authors claimed that bla CTX-M-55 is located on chromosomes or plasmids in 14 strains. Did they do Pulsed-field gel electrophoresis with S1 nuclease and Southern blotting experiments? How else would they select three representative strains for Nanopore sequencing? Please provide the detail information and data.
(2) Line 271-273: This point here is not appropriate. How could you get the point?
There have no IS26 in plasmid IncHI2.
(3) The Figures in this manuscript are too simple and crude to contain much useful information, and are poorly made.
Minor comments: (1) In line 32,33, 36, 37, there should be Spaces between words (2) In line 128, the FastQC reference may not be quite correct, please double-check it.
(3) In lines 131-132, what are the parameters in the process of determining resistance genes and plasmid replication subtypes through the CGE server, for similarity and coverage, etc.? These should be stated.
(4) In line 683, 684, 686 and 687. ISECP1, E.coli, Salmonella as well as resistance genes should be italicized. Such errors also appeared elsewhere in the manuscript, please check.

Response to Reviewers
Thanks all the reviewers for these precious comments and suggestions.
According to reviewer's comments, we answered the questions one by one and showed the revision as follows:

Editor comments:
The focus, clarity, and presentation of the manuscript needs to be improved.
Please expand on the background information provided in the Introduction and ensure the methods are described fully so they can be reproduced by others. The link below provides information on language editing services for your consideration. If these items in addition to the points below are properly addressed, I will consider the publication of this manuscript.

Authors' response:
Thank you for your time for the review of this article. We ensure that the method has been described fully and the background information in the introduction has been appropriately added (Added in the introduction and highlighted in yellow). In addition, the uploaded manuscript has used the language editing service as you recommended.

Reviewer#1 comments:
To identify the epidemiology and the genetic environment of the rising pathogen CTX-M-55 Salmonella in China, authors collected 105 Salmonella strains that include 70 S. Typhimurium. Fourteen Salmonella were carrying blaCTX-M-55 gene 2 S.
Munster, 1 S. Rissen, 1 S. Saintpaul and 10 S. Typhimurium. However, the late 10 strains were phylogenetically divided into 2 close clusters, pointy towards only 2 clones or strains instead of 10 strains. Authors suggested that these clusters should be considered as not detected outbreaks. Considering that, all analysis is based on 2 outbreak strains, all prevalence and epidemiological analysis do not reflect the real epidemiology.
In a published study (2020), not cited in this publication, authors investigated the prevalence of blaCTX-M-55 in 4724 strains in China, concluding to the same finding as this study regarding the location, real prevalence and ST typing.

Authors' response:
Thank you for the suggestion. According to your suggestion, we added the content of "Considering the relatively few of strains in this study, we will continue to collect clinical isolates for follow-up research." in lines 420-421 and highlighted in yellow. In addition, we have carefully read the literature you recommended (Global clonal spread of mcr-3-carrying MDR ST34 Salmonella enterica serotype Typhimurium and monophasic 1,4,[5],12:i:− variants from clinical isolates), and found that its research object is mcr-3-positive strains. There is no doubt that that is an excellent article, which is worth learning. And we cite it in line 107 and highlighted in yellow.

Reviewer#1 comments:
Authors, should consider publishing this work with another angle, as the number of strains does not reflect the title of the study.

Authors' response:
Thank you for the suggestion. According to the review's advice, we changed the original Title "Prevalence of Chromosomally Located blaCTX-M-55 gene in Salmonella Typhimurium ST34 clonal strains from Guangzhou, China" to "Prevalence of Chromosomally Located blaCTX-M-55 gene in Salmonella Typhimurium ST34 clonal strains from Conghua District of Guangzhou, China".

Reviewer#1 comments:
The text should be revised with regard to orthograph, examples: Line 79: space missing between clinic and (6) Line 112: E. coli should be in italic Line 130: space missing between 29 and "and" Line 132: space missing between server and (31) Line 133: 2 spaces between for and subsequent

Authors' response:
Thank you for the suggestion. We carefully checked to ensure that the above errors have been corrected.

Reviewer#2 comments:
The authors claimed that blaCTX-M-55 is located on chromosomes or plasmids in 14 strains. Did they do Pulsed-field gel electrophoresis with S1 nuclease and Southern blotting experiments? How else would they select three representative strains for Nanopore sequencing? Please provide the detail information and data.

Authors' response:
Thank you for the suggestion. According to your advice, we have shown the selection details on the Supplementary Table S1 ( Moreover, both are Cluster 2; Six strains of S. Typhimurium were without resistant plasmid replicates (S24, S34, S36, S42, S49, S79, and S133), belonging to Cluster 1; Even if S. Typhimurium S92 carries IncQ1, the cgMLST results show that it also belongs to Cluster 1; Long-read sequencing of S. Rissen and S. Muenster was not performed due to limited funding. Taken together, we selected S25, S29 and S34 with blaCTX-M-55 gene localized on IncHI2, IncI1 and chromosomes, respectively, as representative strains.

Reviewer#2 comments:
Line 271-273: This point here is not appropriate. How could you get the point?
There have no IS26 in plasmid IncHI2.

Authors' response:
Thank you for the suggestion. In our manuscript, we want to express IS26 at both ends of the genetic framework on the chromosome, not at the ends of the InHI2 plasmid. Sorry for misleading you, so we reorganized content and changed it to "The genetic environment of the IncHI2 is similar to that of the chromosome, the main difference being that IS26 is located at both ends of the chromosome, and ISEcp1 is truncated by an IS26. ISEcp1 upstream of the blaCTX-M-55 gene on IncHI2 was complete ( Fig. 4)", which in line 285-288 now and highlighted in yellow.

Reviewer#2 comments:
The Figures in this manuscript are too simple and crude to contain much useful information, and are poorly made.

Authors' response:
Thank you for the suggestion. According to your suggestion, we have made corresponding changes in line 219-231, 705-712, and figure 3, which highlighted in yellow.

Reviewer#2 comments:
In line 32,33, 36, 37, there should be Spaces between words; In line 683, 684, 686 and 687. ISECP1, E.coli, Salmonella as well as resistance genes should be italicized. Such errors also appeared elsewhere in the manuscript, please check.

Authors' response:
Thank you for the suggestion. We carefully checked to ensure that the above errors have been corrected.

Reviewer#2 comments:
In line 128, the FastQC reference may not be quite correct, please double-check it.

Authors' response:
Thank you for the suggestion. It has been changed in line 136 and highlighted in yellow.

Reviewer#2 comments:
In lines 131-132, what are the parameters in the process of determining resistance genes and plasmid replication subtypes through the CGE server, for similarity and coverage, etc.? These should be stated.

Authors' response:
Thank you for the suggestion. It has been changed in line 140-142 and highlighted in yellow. The sentence added is "CGE's ResFinder 4.1 and PlasmidFinder 2.1 web tools with default settings, threshold for minimum % identity 90% and minimum % coverage 60%, were used for analyses."

Reviewer#3 comments:
This paper characterized 105 clinical Salmonella strains, the results should be more concentrate. For example, how many patients are the strains from? What's the meaning of the result of months and detection rate of male and female patients? This paper can be more important by just describe and discuss most important findings.

Authors' response:
Thank you for the suggestion. A total of 105 NTS non-repetitive strains were isolated from different patients in the Fifth Affiliated Hospital of Southern Medical University, which in line 117-119 and highlighted in yellow. We have added relevant contents in the discussion section; it has been added in line 313-318 and highlighted in yellow.

Reviewer#3 comments:
1.Capitalize the first letter of the title? 2.Spaces are missing in many places 3.Line 112, the name of strain should be in Italic.

Authors' response:
Thank you for the suggestion. We carefully checked to ensure that the above errors have been corrected.

Reviewer#3 comments:
The results should be re-organized. It is better to have a separate result for antibiotic resistance genes in Line 200-207.

Authors' response:
Thank you for the suggestion. Considering that our research object is the blaCTX-M-55 gene, the distribution of other β-lactamase coding genes is shown in the Supplementary Table S2. The position in the re-uploaded manuscript is Line 204-211.

Reviewer#3 comments:
Phylogenetic analysis should contain more strains from China.

Authors' response:
Thank you for the suggestion. According to your suggestion, we downloaded other Chinese Salmonella genome data from EnteroBase, and modified the phylogenetic tree content accordingly, which changes in line 219-231, 705-712, and figure 3, and highlighted in yellow.

Authors' response:
Thank you for the suggestion. The manuscript uploaded again has used the language editing service.

Reviewer#3 comments:
Figure should be more clear.